PROJECT SUMMARY The intracellular, large conductance intracellular RyR calcium release channel is a major regulator of calcium homeostasis in skeletal and cardiac muscle cells. Naturally occurring mutations in the skeletal isoform RyR1 lead to several myopathies (central core disease, multi-minicore disease, nemaline myopathy, exertional rhabdomyolysis) and malignant hyperthermia, whereas mutations in the cardiac isoform RyR2 lead to arrhythmia and heart failure. Some post-translational modifications can also cause similar effects in wild type RyR1 and RyR2. The vast majority of these RyR alterations cause disruption of calcium homeostasis through RyR hypersensitization, which causes sub- threshold opening and sarcoplasmic reticulum calcium leak. The project applies state-of-the-art structural biology technology to determine RyR's structure and allosterism at the atomic level and in a near-native state, in order to examine the molecular mechanism of RyR-mediated calcium leak and analyze how similar modifications affect differently the two RyR isoforms. We will determine the atomic structure of selected RyR1 and RyR2 mutants, and wt RyR1 or RyR2 with post-translational modifications, using cryoEM and 3D image reconstruction. Reaching atomic resolution of non-crystalline samples by cryoEM is now possible owing to the recent development of direct electron detectors for electron microscopy. To further establish and characterize possible heterogeneity in the dataset, classification and multivariate statistical analysis of several thousand RyR cryoEM particles will be performed. Mutant RyRs will be purified from HEK cell lines, and post-translational modifications will be studied on RyR purified from rabbit. Solving the atomic structure of RyR with disease-related alterations will help to understand the molecular mechanism of defective channel closure, and reveal any differential mechanisms between the skeletal and cardiac isoforms. This will advance the field forward towards structure-based, isoform-specific drug design.